DESCRIPTION: The structure of replication origins in the budding yeast S. cerevisiae is relatively well understood. Such origins contain a binding site for the six polypeptide Origin Recognition Complex (ORC) and for one or two additional proteins important for the recruitment of other factors and/or for DNA unwinding. ORC binding appears necessary for the subsequent binding of Cdc6p, the Mcm family of proteins, and perhaps other proteins at the beginning of G1 phase. At the G1/S interface, replication initiates by an unknown mechanism, accompanied by dissociation of Cdc6p and the Mcm family. Homologs to the ORC and Mcm proteins have been found in plants, animals, and other fungi, suggesting that the basic mechanism of initiation is conserved in all eukaryotic organisms. Nevertheless, origins in other tested organisms are less well defined than in S. cerevisiae. For example, compared to S. cerevisiae, there is relaxed specificity for replication origins in the fission yeast S. pombe. In addition, S. pombe origins are more than four-fold larger than those of S. cerevisiae and have more contributing sequence elements. The goal of the proposed studies is to identify proteins that interact with S. pombe origins and compare them to those in S. cerevisiae. This will enable the PI to determine if initiation at S. pombe origins requires new types of proteins as well as ones similar to those in S. cerevisiae. To do this the PI will use linker substitution mutagenesis and in vivo footprinting to accurately define sequences important for two independent S. pombe origins. Proteins that interact with these important sequences also will be sought by genetic screening and (in collaboration with Jerard Hurwitz) by in vitro binding assays.